A Benzoic Acid Terpyridine-Based Cyclometalated Iridium(Iii) Complex As a Two-Photon Fluorescence Probe for Imaging Nuclear Hist

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A benzoic acid terpyridine-based cyclometalated iridium(III) complex as a two-photon fluorescence Cite this: Chem. Commun., 2018, 54, 3771 probe for imaging nuclear histidine†

Received 2nd February 2018, a a ab c d Accepted 14th March 2018 Qiong Zhang, ‡ Xin Lu, ‡ Hui Wang, ‡ Xiaohe Tian, * Aidong Wang, Hongping Zhou, a Jieying Wu a and Yupeng Tian *ae DOI: 10.1039/c8cc00908b

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A series of two-photon active cyclometalated iridium(III) complexes include reduced photobleaching, autofluorescence, non-invasive (Ir1, Ir2 and Ir3) were designed. Ir1 with a two-photon action cross- excitation, and deeper tissue penetration.8–10 However, two- section of 40 GM in the NIR region has been developed for photon absorption (2PA) materials with moderate two-photon targeting intracellular histidine. Two-photon micrographs showed absorption cross-section (s) values usually possess extended that Ir1 could rapidly and selectively light up the nucleus in both p-conjugated planar structures, consequently leading to the use fixed and live cells and is capable of displaying nuclear histidine of biologically unfriendly solvents (e.g. DMSO). Therefore, the distribution in ultra-detail using a super resolution (SR) technique design and synthesis of novel fluorescent probes with histidine under stimulated emission depletion (STED) microscopy. specificity still remain a challenge.11 Recently, the use of precious-metal complexes such as Ru(II), Among the twenty amino acids, histidine is active in maintaining Ir(III) and Pt(II) as luminescent probes has attracted increasing healthy tissues and protecting nerve cells that transport messages interest due to their advantageous photophysical properties and 1,2 12 from the brain to the various other parts of the body. While in low toxicity. Among them, cyclometalated iridium(III)complexes living cells, histidine acts as a structural protein that closely have gained increasing attention in the development of sub- associates with DNA and is known to lead to a variety of ailments cellular location agents or bio-probes due to their unique such as asthma and advanced liver cirrhosis.3 Hence the develop- photophysical properties including large Stokes shifts and high 13 ment of high-quality methods for histidine detection is extremely photostability. To date, iridium(III)-complexes for detecting necessary. Numerous studies have dealt with the detection histidine (His) have rarely been reported.14 And most of these 4 of histidine using techniques such as voltammetry, UV/vis cyclometalated iridium(III) complexes only have a tendency to spectroscopy,5 luminescence spectroscopy methods6 and fluores- be localized in mitochondria.15 In this work, we report a series of 7 cence spectroscopy. However, most of the available probes cyclometalated iridium(III)complexes(Ir1–Ir3) for the purpose of exhibit poor selectivity or require sophisticated detection systems nuclear histidine targeting. Considerations including the photo- such as the use of organic solvents. The development of reliable, physical properties and intracellular behaviour are listed as rapid and accurate methods for the determination of histidine is follows: (1) the Ir–C bond, constructed by 2-phenyl pyridine, was still a highly challenging area. At present, visualization of intra- used to stabilize the energy levels of the Ir complexes. Subsequently, cellular histidine under two-photon microscopy (2PM) is an the two-photon absorption (2PA) activity was tuned using terpyridine attractive approach. The advantages of two-photon excitation derivatives, owing to their strong electron-withdrawing ability, moderate p-conjugated system and strong binding affinity 11 a Department of Chemistry, Key Laboratory of Functional Inorganic Material toward most metal ions. (2) Compared to those in our Chemistry of Anhui Province, Anhui University, Hefei 230601, P. R. China. previous work,11,16 the newly introduced carboxylic acid group E-mail: [email protected] can further influence the extent of electron delocalization and b Department of Chemistry, Wannan Medical College, Wuhu 241002, P. R. China c School of Life Science, Anhui University, Hefei 230601, P. R. China. increase the solubility of the complexes, as well as provide good E-mail: [email protected] biocompatibility. Furthermore, the carboxylic acid group might d School of Chemistry and Chemical Engineering, Huangshan University, accelerate penetration of the iridium(III) complex (Ir1) across Huangshan, P. R. China the nuclear membrane and the phenanthroline group with e State Key Laboratory of Coordination Chemistry, Nanjing University, its excellent planarity can connect with histidine. (3) The Nanjing 210093, P. R. China † Electronic supplementary information (ESI) available. CCDC 1585448. For ESI and additional pyridine ring can provide a lone electron pair, which crystallographic data in CIF or other electronic format see DOI: 10.1039/c8cc00908b could easily be protonated and might lead to good water- ‡ These authors contributed equally to this work. solubility. In addition, the pyridine ring possibly forms a

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Scheme 1 The molecular structure of the Ir(III) complex (Ir1). hydrogen bond with histidine to stabilize the molecule in vivo and in vitro (Scheme 1). The synthesis procedures for Ir1–Ir3 areoutlinedinSchemeS1 (ESI†). The crystal structure information reveals that the Fig. 1 (a) Emission spectra of Ir1 (20 mM) with various amino acids [such iridium(III) centre in Ir2 adopts a distorted octahedral geometry as Ala, Arg, Asn, Gln, Glu, Gly, GSH, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, (Scheme S1, ESI†). One of the pyridine rings was not coordi- Trp, Tyr, Val, Cys, Hcy], ct-DNA, RNA and BSA in PBS buﬀer (pH = 7.4). (b) Changes in the luminescence emission spectra of Ir1 (20 mM) with nated to the Ir1(III) centre indicating that the extra free pyridine 11a,16 various amounts of histidine (0–14 equiv.) in PBS buffer. (c) Two-photon moiety is prone to targeting subcellular organelles. The action cross sections of Ir1 in the presence or absence of His in PBS buffer. linkage between the two planes was conjugated with bond (d) The frontier molecular orbital distributions of Ir1-His. (e) The models lengths of 1.439 Å (C00B–C00H) and 1.452 Å (C00S–C019) for obtained after molecular modeling of the interaction of Ir1 with His. Ir2, revealing that the bond length of C–C is located between that of a normal CQC double bond (1.32 Å) and a C–C single bond (1.53 Å). Notably, there is an active hydrogen atom prone 593 nm when it is excited at 405 nm. The interactions of Ir1 to dissociation and a higher degree of electron delocalization in with numerous intracellular substances including amino acids, the carboxyl group (Fig. S7, ESI†), which caused a nonlinear proteins, DNA and RNA were examined by one and two-photon optical response,17 as well as it maybe being sensitive to amino fluorescence experiments. Only histidine and BSA (a histidine- acids in living cells. The triplet Ir(III) complexes for use in two- rich protein) triggered a significant luminescence enhancement. photon processes require efficient intersystem crossing (ISC), Upon addition of increased concentrations of histidine (Fig. 1(a) large Stokes shifts and relatively long-lived triplet excited state and (b)), a new emission at 493 nm appeared and the intensity lifetimes.18,19 The band at 475 nm is assigned to a p*(C–N) - increase corresponded to a blue shift of 100 nm. The phosphor- p*(L) ligand-to-ligand charge-transfer (LLCT) transition, prob- escence intensity of Ir1 at 493 nm (F o 1%, t = 37.55 ns) was ably mixed with some 3MLCT contribution (Fig. S4(a), ESI†). enhanced up to 27-fold when the concentration of histidine was The temperature-dependent shifts of the emission are typical of increased from 0 to 280 mM(F = 4.81%, t = 97.88 ns). The MLCT phosphorescence (Fig. S4(b), ESI†), arising from the significant enhancement of the phosphorescence quantum yield different environments surrounding the molecule associated suggests that Ir1 is suitable for use in aqueous media. These with stabilization of the polar MLCT emissive state. To support results indicate that Ir1 displays high selectivity for histidine/ the experimentally determined photophysical properties, TDDFT histidine-containing proteins in vitro. To explore the potential calculations showed that the lowest-lying triplet state (T1)is of Ir1 for 2PFM applications, the histidine induced obvious mainly described by the HOMO - LUMO excitation and has a enhancement of the 2PEF of Ir1, corresponding to that of one- 3 3 MLCT/ LLCT nature. The second lowest state (T2), which also has photon excited fluorescence (1PEF) (Fig. S12, ESI†), was inves- 3 3 a MLCT/ LLCT nature, appears B0.3 eV higher in energy than T1 tigated. In addition, two-photon absorption action cross sections and presents some 3LC character due to excitations centred on the (Fs) were further calculated to evaluate the two-photon activity ligand. These transitions are in keeping with the experimental of Ir1 and Ir-His (Fig. 1(c) and Fig. S12, ESI†). The Fs of Ir1 at results, which are attributed to two classes of transition (Fig. S4(c) 780 nm increased from 40 GM to 48 GM (Ir1-His). This result and Table S2, ESI†). indicates that Ir1 undergoes distinct changes in two-photon

The 2PA action cross-section (dmax)ofIr1 in DMSO/water activity before and after reacting with His, which implies that (DMSO : H2O = 1 : 9) is presented in Fig. S4(d) (ESI†) and Ir1 is suitable for tracking histidine using the 2PFM technique. Fig. 1(c). The largest 2PA action cross-sections of Ir complexes To verify the above sensing mechanism, 1H NMR titration were located around 780 Æ 20 nm with dmax values between experiments were carried out in d6-DMSO : D2O = 9 : 1 (v/v).

20 and 40 GM. The intensities of 2PEF clearly exhibit the sequence A significant peak split of the hydroxy protons Ha and Hb Ir1 4 Ir3 4Ir2 4 L. Consistent with the above discussion, occurred after the addition of 2 equiv. of histidine into the we found that a moderate extended bridge can be reasonably Ir1 solution, demonstrating the presence of secondary bond inter- increased by the length of the conjugated chain and this is actions between the carboxyl group of Ir1 and the imidazole group more favourable to electronic transition floating, which is in of histidine. The prominent peak at m/z 1009.2513 corresponding agreement with the increase of the 2PA cross-section. to the product [Ir1-His] was found using HRMS (Fig. S13, ESI†). In order to evaluate their application in vivo and in vitro, The calculated frontier molecular orbitals and the calculated data Ir1 was selected due to it having the largest 2PA cross-section for Ir1 and Ir1-His are shown in Fig. 1(d) and Table S3 (ESI†). For (Fig. S4(d), ESI†). Ir1 exhibits a weak emission band at about Ir1-His, the triplet excitations corresponding to the triplet–triplet

3772 | Chem. Commun., 2018, 54, 3771--3774 This journal is © The Royal Society of Chemistry 2018 ChemComm Communication transitions (498.5 nm) mainly originate from the HOMO - LUMO+1 transition. The distribution of LUMO+1 is similar to that of the LUMO of Ir1 and is mainly localized on the whole terpyridine ligand. However, the distribution of the HOMO of Ir1-His is significantly different from that of Ir1, which resides on the imidazole part of histidine. Thus, an admixture of 3 3 [dp(Ir) - p*terpyridine] MLCT and [pimidazole - p*terpyridine] 3LLCT is responsible for the triplet–triplet transitions of Ir1-His, improving the emission of the iridium(III) complex. Therefore, the significant enhancement in the emission intensity of Ir1-His may arise from a less nonradiative process and a favorable ICT process in the rigid molecular structure and the variation in transition character. The mechanism was further confirmed via molecular modelling calculations using Discovery Studio Software.20 The docking results (Fig. 1(e)) indicate that Ir1 with a suitable positive Fig. 2 (a) Determination of intracellular localization of Ir1 by confocal charge and lipophilicity, which readily triggers histidine through two-photon microscopy. HepG2 cells were incubated with Ir1 (10 mM, lex = 820 nm, lem = 580–620 nm) for 10 min and then co-incubated with secondary bond interactions in different directions, can be stable EM NucRed (lex =638nm,lem = 686 nm) for 10 min at 37 1C(inset:Pearson’s with a high dock score and low CDOCKER energy. coefficients Rr = 0.97), scale bar = 20 mM. (b (inset)) Amplified confocal two- To further explore their 2PM applications, the MTT results photon fluorescence imaging of living HepG2 cells incubated with 10 mM Ir1. for Ir1 demonstrated the relatively low toxicity of the complex (c (inset)) Amplified confocal two-photon fluorescence imaging of fixed (Fig. S14, ESI†). The photostability of Ir1 was shown by almost no HepG2 cells incubated with 10 mM Ir1. (d) Two-photon fluorescence intensity profile across the line shown in yellow corresponding to the obvious luminescence change being observed during B250 s of extracellular region (nucleus and cytoplasm). (e) Two-photon fluorescence continued irradiation (Fig. S15, ESI†). This indicated that the images of Ir1 in HepG2 cells: HepG2 cells incubated with Ir1 (10 mM) for high photostability of Ir1 makes it favourable for long-term real- 30 min. (f) HepG2 cells pretreated with histidine decarboxylase (1.0 mM) time tracking. We used HepG2 cells as a cell model incubated for 30 min and then incubated with Ir1 (10 mM) for 30 min. (g) HepG2 cells with Ir1; the colocalization experiments using commercially pretreated with histidine decarboxylase (1.0 mM) for 30 min and then incubated with histidine (400 mM) for 1 h, and finally incubated with Ir1 available nuclear stain Nuclear Reds (Fig. 2(a)) strongly sug- (10 mM) for 1 h. (h) Cellular localization of Ir1 characterized by transmission gested that Ir1 targets the cell nucleus in living cells leading to electron microscopy. TEM image of HepG2 cells incubated with Ir1 and high signal-to-noise ratios (Fig. 2(d)). Inductively coupled plasma stained without osmium tetroxide. (i) TEM image of HepG2 cells incubated with (ICP)-MS was carried out to verify our speculations; the majority the complex Ir1 stained without osmium tetroxide (right) (scale bar = 5 mm). of iridium was localized and accumulated in the nucleus fraction of the cells which indicated a ‘‘turn on’’ effect in the nucleus (Fig. S16, ESI†). Similar nuclear uptake was displayed on appli- that has been widely used in electron microscopy, in the treated cation in other types of cancerous cell (HeLa and A549) (Fig. S17, cells, subcellular membrane structures were clearly observed, ESI†). Therefore, it was concluded that Ir1 can selectively stain including mitochondria, intracellular vesicles, plasma mem- the nuclear region in various cancer cells. Moreover, specific branes and nuclear membranes. It was found that the cell nucleus staining by Ir1 was achieved only for living cells and not nucleus displayed much better contrast due to the binding of for fixed cells. A generalized diffuse whole-cell staining pattern Ir1 with nuclear histidine (Fig. 2(i)) when Ir1 was used as the was observed by 2PM (Fig. 2(b) and (c)) when HepG2 cells were sole contrast agent. These findings are again in agreement with fixed and further incubated with Ir1. Notably the normal cells, the 2PM imaging, all strongly suggesting that Ir1 was located HELF cells (Human Embryo Liver Fibroblast), displayed diffusion within the nucleus in live cells with histidine specificity. of Ir1 throughout the cell cytosolic region and were excluded The cellular entry pathway of Ir1 was also investigated as from the cell nucleus (Fig. S18, ESI†), which might suggest that displayed in Fig. S19 (ESI†). It shows that no luminescence was the nuclear uptake pattern of Ir1 was cancer specific, however its observed at 4 1C, indicating that Ir1 might enter cells through a actual mechanism is still under investigation. temperature-dependent pathway. Along with Ir1 having been To further confirm that the binding substance of Ir1 in the successfully utilized as a convenient and noninvasive 2PA probe living cell nucleus is indeed histidine, histidine decarboxylase in living cells with superb photostability and cell permeability, (HDC) that can be reacted with histidine species was used for it also showed good deep tissue penetration on a solid tumor pre-incubation with HepG2 cells. HepG2 cells showed a lumi- model using HepG2 cells and spherical multi-cells and dis- nescence off–on effect (Fig. 2g) after histidine + Ir1 treatment, played nuclear labeling. A significant fluorescence intensity was strongly suggesting that Ir1 is capable of targeting intracellular observed from the surface of spheroids to a depth of B66 mm nuclear histidine with high specificity. Such a conclusion was for Ir1 (Fig. S20, ESI†), indicating deep penetration of the two- further proved by transmission electron microscopy experiments photon excitation light. using Ir1 due to its high electron density and intracellular in situ The successful nuclear histidine labeling using Ir1 demon- accumulation. Compared to control cells stained solely with strated stable phosphorescence, high specificity, a large Stokes osmium tetroxide (Fig. 2(h)), a phospholipid contrast reagent shift and a long fluorescence lifetime (Table S1, ESI†), so we were

Anhui Province (2016B092), and the China Postdoctoral Science Foundation (2017M612050), the Higher Education Revitalization Plan Talent Project (2013).

Conflicts of interest

There are no conflicts to declare.

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